Methods and compositions for modulating peripheral immune function

ABSTRACT

Disclosed herein are cell preparations useful for modulating various peripheral immune functions, methods for making said cell preparations, and methods for their use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/298,001 filed Jun. 6, 2014 (now U.S. Pat. No. 9,655,927 issued May23, 2017), which is a continuation of U.S. patent application Ser. No.13/441,311 filed Apr. 6, 2012 (now U.S. Pat. No. 8,785,190 issued Jul.22, 2014), which claims the benefit of priority to U.S. ProvisionalPatent Application No. 61/516,637 filed Apr. 6, 2011 and 61/541,248filed Sep. 30, 2011, the contents of which are incorporated herein byreference in their entireties.

STATEMENT REGARDING FEDERALLY SUPPORT

Not applicable

FIELD

The disclosure is in the field of immunomodulation (e.g.,immunosuppression).

BACKGROUND

Peripheral (i.e., non-CNS) immunity in vertebrates is mediated by twosystems: the innate immune system and the adaptive immune system. Theinnate immune system provides an early, non-specific response to injuryand/or infection. By contrast, the adaptive immune system is broughtinto play later in the process of injury or infection, and is specificto the invading pathogen. The innate immune system, being evolutionarilymore ancient, is active in plants, invertebrates and vertebrates, whilethe adaptive immune system is active in vertebrates only.

As noted above, the innate immune system becomes active immediately uponinfection, at the site of infection, and does not depend on priorexposure to the infecting pathogen. It thus provides a set of generaldefense mechanisms that are not specific to any particular pathogen.Cellular elements of the innate immune system include macrophages,dendritic cells, neutrophils and natural killer (NK) cells.Macromolecular components of the innate immune system include defensinpeptides and the complement system. Additional elements of innateimmunity include physical barriers to infection (such as thekeratinization of the skin, tight junctions between epithelial cells,stomach acid and the mucus secreted by many epithelial tissues) andcell-intrinsic responses such as, for example, phagocytosis (sometimescoupled with lysosomal fusion of phagocytosed material) and degradationof double-stranded RNA.

Activation of the innate immune system is mediated, in part, byrecognition of pathogen-associated molecules such as, for example,N-formyl methionine-containing polypeptides, cell wall peptidoglycans,bacterial flagella, lipopolysaccharides, techoic acid, andfungal-specific molecules such as mannan, glucan and chitin. Inaddition, certain nucleic acid sequences common to microorganisms (suchas unmethylated CpG dinucleotides) can trigger innate immune responses.Recognition of such pathogen-associated immunostimulants results in themounting of an inflammatory response and phagocytosis of the pathogen bymacrophages, neutrophils and/or dendritic cells.

Certain of the pathogen-associated immunostimulants noted above occur inrepeating patterns called pathogen-associated molecular patterns(PAMPs), which can be recognized by pattern recognition receptors on thesurfaces of innate immune system cells. These receptors include solublemembers of the complement system and membrane-bound receptors such asmembers of the Toll-like receptor family (TLRs) and the so-called NODproteins. The membrane-bound receptors can stimulate phagocytosis andactivate programs of gene expression responsible for various innate andadaptive immune responses.

Finally, the innate immune system is involved in activating adaptiveimmunity, in part by secreting extracellular signaling molecules whichstimulate proliferation and differentiation of cells of the adaptiveimmune system, and also by processing and presenting antigens to cellsof the adaptive immune system.

The adaptive immune system, in contrast to the innate immune system, isnot activated immediately upon infection, and generates specific,long-lived responses to pathogens. Activation of the adaptive immunesystem occurs not at the site of injury, but in lymphoid organs, anddepends on presentation of antigens by components of the innate immunesystem to activate cells of the adaptive immune system. The principalcells of the adaptive immune system are B-lymphocytes (B cells), whichsynthesize and secrete antibodies, and T-lymphocytes (T cells).

There are three major classes of T cells: cytotoxic, helper, andregulatory (or suppressor) T cells. Cytotoxic T cells are able to killinfected host cells. Helper T cells participate in activation ofmacrophages, dendritic cells, B cells and cytotoxic T cells by secretingcytokines and/or by surface expression of one of a number of differentco-stimulatory molecules. There are two types of helper T cells: T_(H)1cells participate in activation of macrophages, cytotoxic T cells and Bcells to provide immunity to intracellular pathogens and secrete themacrophage-activating cytokines interferon gamma (IFN-γ) and tumornecrosis factor alpha (TNF−α). T_(H)1 cells are also capable ofstimulating inflammatory responses. T_(H)2 cells help activate B cellsto produce antibodies, primarily in response to extracellular pathogens,and secrete the cytokines interleukin 4 (IL4) and interleukin 10 (IL10).Development of a naive helper T cell into a T_(H)1 cell is stimulated byinterleukin 12 (IL12); while pathogen-induced expression of the Jaggedprotein by a dendritic cell will guide a naive helper T cell to developinto a T_(H)2 cell producing IL4, which stimulates antibody productionby B cells. Regulatory T cells (T_(reg)s) inhibit the function ofcytotoxic T cells, helper T cells and dendritic cells, and are unique inexpressing the Foxp3 transcription factor. Thus, the interplay betweenhelper T cells and regulatory T cells helps keep the immune response inbalance, with sufficient activity to clear an invading pathogen withoutexcessive damage to the host.

A class of lymphocytes in the adaptive immune system known as memorycells retains receptors to a pathogen subsequent to infection andclearance, enabling the host organism to mount a more rapid adaptiveimmunological response to a subsequent encounter with the same pathogen,and providing the basis for natural or vaccination-induced immunity tomany infections diseases. By contrast, the innate immune system does notretain such immunological memory.

Mesenchymal stem cells (MSCs, also known as “marrow stromal cells” or“marrow adherent stem cells”), that have been transfected with a plasmidexpressing the Notch intracellular domain (NICD), are useful for thetreatment of a number of diseases and disorders of the central andperipheral nervous systems. See, for example, U.S. Pat. No. 7,682,825(Mar. 23, 2010); US Patent Application Publication No. 2006/0216276(Sep. 28, 2006); US Patent Application Publication No. 2010/0034790(Feb. 11, 2010) US Patent Application Publication No. 2010/0310523 (Dec.9, 2010); International Patent Application Publication No. WO 08/102460(Aug. 28, 2008); Yasuhara et al. (2009) Stem Cells and Development18:1501-1513 and Glavaski-Joksimovic et al. (2009) Cell Transplantation18:801-814.

The ability of these cells, known as SB623 cells, to rescue damagedneural tissue is associated, in part, with their secretion of varioustrophic factors and their elaboration of various extracellular matrixcomponents. See, for example, US Patent Application Publication No.2010/0266554 (Oct. 21, 2010) and US Patent Application Publication No.2010/0310529 (Dec. 9, 2010).

Current cell transplantation therapies have significant disadvantages,including, for example, host peripheral immunological reactions to thetransplanted cells. In addition, inflammation is a hallmark of manyneurodegenerative diseases, such as, for example, Parkinson's diseaseand multiple sclerosis. Villoslada et al. (2008) Clin. Immunol.128:294-305. MSCs have been reported to attenuate peripheral immuneactivity through mechanisms that include blocking production ofantigen-presenting cells and altering the cytokine profile of helperT-cells. Kong et al. (2009) J. Neuroimmunol. 207:83-91. However, MSCshave limited regenerative potential, becoming senescent following exvivo manipulation. Wagner et al. (2008) PLoS One 3:e2213; Jin et al.(2010) Biochem Biophys Res Commun. 391:1471-1476. Although senescentcells secrete a number of cytokines which could be beneficial for tissueregeneration, the overall senescent cell secretory profile ispro-inflammatory. Rodier et al. (2009) Nature Cell Biol. 11:973-979:Coppe et al. (2008) PLoS Biol. 6:2853-2868; Freund et al. (2010) TrendsMol. Med. 16(5):238-246.

For these and other reasons, there remains a need for methods andcompositions for cell transplantation that do not provoke hostperipheral immune responses, and/or that reduce inflammatory, and otherimmune, responses.

SUMMARY

The inventors have identified, within cultures of MSCs that have beentransfected with sequences encoding a Notch intracellular domain andtheir descendants (i.e., SB623 cells), a population of senescent cells.Although SB623 cells have been shown to be capable of treating a numberof central nervous system disorders, the present application disclosesthe surprising ability of SB623 cells to modulate a number of peripheralimmune functions. For example, SB623 cells can inhibit human T cellproliferation in both allogeneic and xenogeneic mixed lymphocytereactions, stimulate IL-10 production by T-cells, and block thedifferentiation of monocytes to dendritic cells. SB623 cells alsoinhibit maturation of dendritic cells and, compared to the parentalMSCs. SB623 cells exert a greater inhibitory effect on dendritic cellmaturation, as evidenced by greater reduction in the surface expressionof the co-stimulatory molecule, CD86. SB623 cells can also convert thecytokine profile of a T-cell population from one that ispro-inflammatory to one that is anti-inflammatory. These properties ofSB623 cells are additionally surprising and unexpected in light ofstudies reporting that senescent cells secrete pro-inflammatorycytokines. Orjalo et al. (2009) Proc. Natl. Acad. Sci. USA106:17031-17036.

Accordingly, SB623 cells, and/or their subpopulation of senescent cells,are useful in a number of therapeutic methods, as exemplified in thefollowing embodiments.

1. A method for peripheral immunosuppression in a subject, the methodcomprising administering to the subject an effective amount of SB623cells; wherein said SB623 cells are obtained by (a) providing a cultureof mesenchymal stem cells; (b) contacting the cell culture of step (a)with a polynucleotide comprising sequences encoding a Notchintracellular domain (NICD) wherein said polynucleotide does not encodea full-length Notch protein; (c) selecting cells that comprise thepolynucleotide of step (b); and (d) further culturing the selected cellsof step (c) in the absence of selection.

2. A method for inhibiting a peripheral inflammatory response in asubject, the method comprising administering to the subject an effectiveamount of SB623 cells wherein said SB623 cells are obtained by (a)providing a culture of mesenchymal stem cells; (b) contacting the cellculture of step (a) with a polynucleotide comprising sequences encodinga Notch intracellular domain (NICD) wherein said polynucleotide does notencode a full-length Notch protein; (c) selecting cells that comprisethe polynucleotide of step (b); and (d) further culturing the selectedcells of step (c) in the absence of selection.

3. The method of embodiment 2, wherein the peripheral inflammatoryresponse results from an allogeneic transplantation, ischemia ornecrosis.

4. A method for suppressing peripheral T-cell activation in a subject;the method comprising administering to the subject an effective amountof SB623 cells; wherein said SB623 cells are obtained by (a) providing aculture of mesenchymal stem cells; (b) contacting the cell culture ofstep (a) with a polynucleotide comprising sequences encoding a Notchintracellular domain (NICD) wherein said polynucleotide does not encodea full-length Notch protein; (c) selecting cells that comprise thepolynucleotide of step (b); and (d) further culturing the selected cellsof step (c) in the absence of selection.

5. The method of embodiment 4, wherein said peripheral T-cell activationcomprises expression of CD69 and/or HLA-DR by the T-cells.

6. The method of embodiment 4, wherein said peripheral T-cell activationcomprises proliferation of CD4⁺ T-cells.

7. A method for suppressing the function of peripheral helper T-cells ina subject; the method comprising administering to the subject aneffective amount of SB623 cells; wherein said SB623 cells are obtainedby (a) providing a culture of mesenchymal stem cells; (b) contacting thecell culture of step (a) with a polynucleotide comprising sequencesencoding a Notch intracellular domain (NICD) wherein said polynucleotidedoes not encode a full-length Notch protein; (c) selecting cells thatcomprise the polynucleotide of step (b); and (d) further culturing theselected cells of step (c) in the absence of selection.

8. The method of embodiment 7, wherein said peripheral helper T-cellfunction is cytokine secretion.

9. The method of embodiment 7, wherein said peripheral helper T-cellfunction is associated with the pathology of rheumatoid arthritis.

10. A method for expanding a population of peripheral regulatory T-cells(T_(reg)s) in a subject; the method comprising administering to thesubject an effective amount of SB623 cells; wherein said SB623 cells areobtained by (a) providing a culture of mesenchymal stem cells; (b)contacting the cell culture of step (a) with a polynucleotide comprisingsequences encoding a Notch intracellular domain (NICD) wherein saidpolynucleotide does not encode a full-length Notch protein; (c)selecting cells that comprise the polynucleotide of step (b); and (d)further culturing the selected cells of step (c) in the absence ofselection.

11. A method for modulating peripheral production of a cytokine in asubject; the method comprising administering to the subject an effectiveamount of SB623 cells; wherein said SB623 cells are obtained by (a)providing a culture of mesenchymal stem cells; (b) contacting the cellculture of step (a) with a polynucleotide comprising sequences encodinga Notch intracellular domain (NICD) wherein said polynucleotide does notencode a full-length Notch protein; (c) selecting cells that comprisethe polynucleotide of step (b); and (d) further culturing the selectedcells of step (c) in the absence of selection.

12. The method of embodiment 11, wherein the cytokine is apro-inflammatory cytokine and production of the cytokine is reduced.

13. The method of embodiment 12, wherein the cytokine is produced by a Tcell.

14. The method of embodiment 13, wherein the cytokine is interferongamma (IFN-γ).

15. The method of embodiment 12, wherein the cytokine is produced by amonocyte.

16. The method of embodiment 15, wherein the cytokine is tumor necrosisfactor-alpha (TNF-α).

17. The method of embodiment 11, wherein the cytokine is ananti-inflammatory cytokine and production of the cytokine is stimulated.

18. The method of embodiment 17, wherein the cytokine is interleukin-10(IL-10).

19. The method of embodiment 18, wherein the cytokine is produced by a Tcell or a monocyte.

20. The method of embodiment 19, wherein the T cell is a helper T-cell.

21. The method of embodiment 20, wherein the helper T-cell is a T_(H)1cell.

22. The method of embodiment 19, wherein the cytokine is produced by aregulatory T-cell.

23. The method of embodiment 22, wherein the regulatory T-cell is aT_(R)1 cell

24. A method for inhibiting the differentiation of a peripheral monocyteto a dendritic cell in a subject; the method comprising administering tothe subject an effective amount of SB623 cells; wherein said SB623 cellsare obtained by (a) providing a culture of mesenchymal stem cells; (b)contacting the cell culture of step (a) with a polynucleotide comprisingsequences encoding a Notch intracellular domain (NICD) wherein saidpolynucleotide does not encode a full-length Notch protein (c) selectingcells that comprise the polynucleotide of step (b); and (d) furtherculturing the selected cells of step (c) in the absence of selection.

25. A method for inhibiting the maturation of a peripheral dendriticcell in a subject; the method comprising administering to the subject aneffective amount of SB623 cells; wherein said SB623 cells are obtainedby (a) providing a culture of mesenchymal stem cells; (b) contacting thecell culture of step (a) with a polynucleotide comprising sequencesencoding a Notch intracellular domain (NICD) wherein said polynucleotidedoes not encode a full-length Notch protein; (c) selecting cells thatcomprise the polynucleotide of step (b); and (d) further culturing theselected cells of step (c) in the absence of selection.

26. The method of embodiment 25, wherein maturation comprises anincrease in expression of CD86 by the dendritic cell.

27. A method for treating GVHD in a subject; the method comprisingadministering to the subject an effective amount of SB623 cells; whereinsaid SB623 cells are obtained by (a) providing a culture of mesenchymalstem cells; (b) contacting the cell culture of step (a) with apolynucleotide comprising sequences encoding a Notch intracellulardomain (NICD) wherein said polynucleotide does not encode a full-lengthNotch protein; (c) selecting cells that comprise the polynucleotide ofstep (b); and (d) further culturing the selected cells of step (c) inthe absence of selection.

28. A method for inhibiting graft rejection in a subject; the methodcomprising administering to the subject an effective amount of SB623cells; wherein said SB623 cells are obtained by (a) providing a cultureof mesenchymal stem cells; (b) contacting the cell culture of step (a)with a polynucleotide comprising sequences encoding a Notchintracellular domain (NICD) wherein said polynucleotide does not encodea full-length Notch protein; (c) selecting cells that comprise thepolynucleotide of step (b); and (d) further culturing the selected cellsof step (c) in the absence of selection.

29. A method for treating a peripheral autoimmune disorder in a subject;the method comprising administering to the subject an effective amountof SB623 cells; wherein said SB623 cells are obtained by (a) providing aculture of mesenchymal stem cells; (b) contacting the cell culture ofstep (a) with a polynucleotide comprising sequences encoding a Notchintracellular domain (NICD) wherein said polynucleotide does not encodea full-length Notch protein; (c) selecting cells that comprise thepolynucleotide of step (b); and (d) further culturing the selected cellsof step (c) in the absence of selection.

30. The method of embodiment 29, wherein the peripheral autoimmunedisorder is selected from the group consisting of multiple sclerosis,ulcerative colitis, chronic obstructive pulmonary disease (COPD),asthma, lupus and Type I diabetes.

31. The method of any of the preceding embodiments, wherein the subjectis an experimental animal.

32. The method of any of embodiments 1-30, wherein the subject is ahuman.

33. A method for modulating a peripheral inflammatory response in asubject, the method comprising administering to the subject an effectiveamount of SB623 cells; wherein said SB623 cells are obtained by:

(a) providing a culture of marrow adherent stromal cells;

(b) contacting the cell culture of step (a) with a polynucleotidecomprising sequences encoding a Notch intracellular domain (NICD)wherein said polynucleotide does not encode a full-length Notch protein:

(c) selecting cells that comprise the polynucleotide of step (b); and

(d) further culturing the selected cells of step (c) in the absence ofselection for the polynucleotide, such that said marrow adherent stromalcells are induced to form SB623 cells by expression of the NICD.

34. The method of embodiment 33, wherein said modulating comprisesinhibition of activation of peripheral T-cells.

35. The method of embodiment 34, wherein said activation of peripheralT-cells comprises expression of CD69 and/or HLA-DR by the T-cells.

36. The method of embodiment 34, wherein said activation of peripheralT-cells comprises proliferation of CD4 T-cells.

37. The method of embodiment 33, wherein said modulating comprisesinhibition of helper T-cell function.

38. The method of embodiment 37, wherein said helper T-cell function iscytokine secretion.

39. The method of embodiment 37, wherein said helper T-cell function isassociated with the pathology of rheumatoid arthritis.

40. The method of embodiment 33, wherein said modulating comprisesexpanding a population of peripheral regulatory T-cells (T_(reg)s).

41. The method of embodiment 33, wherein said modulating comprisesinhibition of differentiation of a monocyte to a dendritic cell.

42. The method of embodiment 33, wherein said modulating comprisesinhibition of the maturation of a dendritic cell.

43. The method of embodiment 42, wherein the maturation comprises anincrease in expression of CD86 by the dendritic cell.

44. The method of embodiment 33, wherein the peripheral inflammatoryresponse results from an allogeneic transplantation, ischemia ornecrosis.

45. The method of embodiment 33, wherein the peripheral inflammatoryresponse results from graft-versus-host disease (GVHD) or graftrejection.

46. The method of embodiment 33, wherein the peripheral inflammatoryresponse results from an autoimmune disorder.

47. The method of embodiment 46, wherein the autoimmune disorder isselected from the group consisting of multiple sclerosis, ulcerativecolitis, chronic obstructive pulmonary disease (COPD), asthma, lupus andType I diabetes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows measurements of CFSE dilution, in MSCs and SB623 cells, byflow cytometry.

FIG. 2 shows changes in cell count in cultures of MSCs and SB623 cells,determined by Trypan Blue exclusion after three days of culture. Eachculture was started with one million cells.

FIGS. 3A and 3B show the results of propidium iodide staining ofcultures of MSCs and SB623 cells. FIG. 3A shows representative FACSdata. The peak labeled “M1” represents resting (G0/G1) phase cells. FIG.3B shows the fraction of cells in the resting phase of the cell cycle,for MSCs and SB623 cells, determined by measuring the area of the M1peak in FIG. 3A.

FIG. 4 shows measurements of p16Ink4A levels in MSCs and SB623 cells.

FIG. 5 shows levels of certain surface markers in MSCs and SB623 cells.

FIG. 6 shows measurements of CD54 expression in MSCs and SB623 cells.

FIG. 7 shows levels of certain cytokines in MSCs and SB623 cells.

FIG. 8 shows levels of transforming growth factor beta-1 (TGF-β-1) andvascular endothelial growth factor-A (VEGF-A) in MSCs and SB623 cells.

FIGS. 9A and 9B, show the effect of SB623 cells and MSCs on T-cellactivation in an allogeneic MLR. FIG. 9A shows representative FACStraces, gating on CFSE and phycoerythrin-labeled anti-CD69, for controlunstimulated human T-cells (upper left panel, indicated by “-”); humanT-cells stimulated by allogeneic PBMCs (upper right panel, indicated by“MLR”); MLR as before with 10⁴ MSCs (lower left panel, indicated by“MLR+MSC”) and MLR as before with 10⁴ SB623 cells (lower right panel,indicated by “MLR+SB623”). FIG. 9B shows quantitation of CD69 expressionin MLR cultures. Control, unstimulated T-cell cultures are representedby “Serum;” PBMC-stimulated T-cells in a mixed lymphocyte reaction arerepresented by “MLR;” a mixed lymphocyte reaction as before in thepresence of mesenchymal stem cells is represented by “MSC;” and a mixedlymphocyte reaction as before in the presence of SB623 cells isrepresented by “SB623.” The values for “MSC” and “SB623” are averages ofthree cultures, each containing MSCs or SB623 cells from differentdonors.

FIGS. 10A and 10B show a comparison of T-cell proliferation rates in anallogeneic MLR, quantitated by measuring CFSE dilution. FIG. 10A showsrepresentative FACS traces for control unstimulated human T-cells (upperleft panel, indicated by “T cells alone”); human T-cells stimulated byallogeneic PBMCs (upper right panel, indicated by “MLR”); MLR as beforewith 10⁴MSCs (lower left panel, indicated by “MLR+MSC”) and MLR asbefore with 10⁴ SB623 cells (lower right panel, indicated by“MLR+SB623”). FIG. 10B shows quantitation of CSFE dilution in MLRcultures. Compositions of the cultures are as indicated in FIG. 10A.

FIG. 11 shows a comparison of HLA-DR expression under different cultureconditions. Control, unstimulated T-cell cultures are represented by“Serum” PBMC-stimulated T-cells in a mixed lymphocyte reaction arerepresented by “MLR;” a mixed lymphocyte reaction in the presence ofmesenchymal stem cells is represented by “MSC;” and a mixed lymphocytereaction in the presence of SB623 cells is represented by “SB623.” Thevalues for “MSC” and “SB623” are averages of three cultures, eachcontaining MSCs or SB623 cells from different donors.

FIG. 12 shows effect of SB623 cells and MSCs on T-cell proliferation ina xenogeneic lymphocyte stimulation reaction. Proliferation was measuredby dilution of PKH26, a cell-permeable dye. The percentage of CD32⁺T-cells containing PKH26 were measured for unstimulated T-cells (“Tcells alone”); T-cells co-cultured with glial mix cells (“xeuo-MLR”);T-cells co-cultured with glial mix cells and mesenchyrnal stem cells(“xeno-MLR+MSC”) and T-cells co-cultured with glial mix cells and SB623cells (“xeno-MLR+SB623”). Preparations of MSCs and SB623 cells wereobtained from three different donors, as indicated in the figure.

FIGS. 13A and 13B show assays for regulatory T-cells (T_(reg)s) in invitro T-cell cultures, using coexpression of CD4 and CD25 as a markerfor T_(reg)s. FIG. 13A shows representative FACS traces, measuring CD4and CD25, for IL-2-stimulated T-cells (“T cells”), and IL-2-stimulatedT-cells co-cultured for seven days with either mesenchymal stem cells(“T cells+MSCs”) or SB623 cells (“T cells+SB623 cells”). FIG. 13B showsmean CD4/CD25 expression levels for 5 different matched lots of MSCs andSB623 cells. Note that, for “Donor 1 PBL” a significant increase inCD4⁺CD25⁺ cells is observed (p<0.05) in the co-culture with SB623 cells,compared to the co-culture with MSCs.

FIGS. 14A and 14B show levels of the FoxP3 transcription factor inT-cells cultured in the presence of IL-2, measured by staining forintracellular FoxP3 with a PE-conjugated anti-FoxP3 antibody, followedby flow cytometry analysis. FIG. 14A shows representative FACS tracesfor T-cells cultured in the absence of IL-2 (indicated by “RPMI/10%FBS”), T-cells cultured in 10 ng/ml IL-2 (indicated “+IL-2”), T-cellscultured in IL-2 as above and co-cultured with MSCs (indicated “+MSC”),and T-cells cultured in IL-2 as above and co-cultured with SB623 cells(indicated “+SB623”).

FIG. 14B shows the mean percentage of FoxP3-expressing T-cells afterculture in the presence of IL-2 (indicated “T cells alone”) or afterco-culture with MSCs (“T cells+MSC”) or SB623 cells (“T cells+SB623”) inthe presence of IL-2. Co-culture was conducted with 3 different matchedlots of MSCs and SB623 cells.

FIGS. 15A and 15B show results of measurements of intracellular IL-10levels in CD4⁺ T-cells cultured in the presence of IL-2. FIG. 15A showsrepresentative FACS traces for T-cells cultured in the presence of IL-2(“T cells alone”), T-cells cultured in IL-2 as above and co-culturedwith MSCs (indicated “T cells+MSC”), and T-cells cultured in IL-2 asabove and co-cultured with SB623 cells (indicated “T cells+SB623”).Alexa 488 fluorescence, indicative of IL-10 levels, is shown on theabscissa. FIG. 15B shows mean percentage of IL-10-positive cells inco-cultures of T-cells with three different matched lots of MSCs (“Tcells+MSC”) and SB623 cells (“T cells+SB623”), compared T-cells thatwere not co-cultured (“T cells alone”).

FIGS. 16A and 16B, show levels of cytokines in T-cells cultured in theabsence of IL-2 and in the presence of non-maximally-inducing levels ofPMA and ionomycin. In FIG. 16A, levels of interferon-gamma (IFN-g) areshown in freshly-isolated T-cells prior to culture (“Fresh cells”),T-cells cultured for seven days in the absence of other cells (“Culturecontrol”), T-cells co-cultured with SB623 cells for seven days(“SB623”), and T-cells co-cultured with MSCs for seven days (“MSC”). InFIG. 16B, levels of interleukin-10 (IL-10) are shown in freshly isolatedT-cells prior to culture (“Fresh cells”), T-cells cultured for sevendays in the absence of other cells (“Culture control”), T-cellsco-cultured for seven days with SB623 cells (“SB623”), and T-cellsco-cultured for seven days with MSCs (“MSC”). The values for “MSC” and“SB623” are averages of three cultures, each containing MSCs or SB623cells from a different donor.

FIG. 17 shows levels of IL-17 in IL-23-stimulated T-cells. Percentexpression was determined by flow cytometry after staining cells forIL-17 with a fluorescent antibody. T-cells were cultured with or withoutIL-23, as indicated, and alone or in co-culture with MSCs or SB623cells, as indicated.

FIGS. 18A and 18B show levels of CD1a and CD14 in monocyte culturesafter 7 days of culture or co-culture. FIG. 18A shows representativeFACS traces of cells stained for CD 1A and CD 14. Monocytes contain apopulation of CD1A^(+CD)14⁺ dendritic cell precursors (leftmost panel).When monocytes were cultured in the presence of IL-4 and GM-CSF for 7days, this dendritic cell precursor population is reduced and replacedby a population of CD1A⁺CD14⁻ dendritic cells (second panel from left).When monocytes are co-cultured with MSCs (third panel from left) orSB623 cells (rightmost panel) in the presence of IL-4 and GM-CSF, theCD1A⁺CD14⁻ dendritic cell population is reduced and the CD1A⁺CD14⁺precursor cell population is increased. FIG. 18B shows mean expressiondata for monocytes (leftmost pair of bars), monocytes cultured in thepresence of IL-4 and GM-CSF for 7 days (second pair of bars from left),monocytes co-cultured with MSCs in the presence of IL-4 and GM-CSF forseven days (third pair of bars from left) or monocytes co-cultured withSB623 cells in the presence of IL-4 and GM-CSF for seven days(right-most pair of bars). Results for the co-culture experiments wereobtained from three different matched lots of MSCs and SB623 cells.

FIG. 19 shows levels of CD86, expressed as mean fluorescent intensity,in TNF-α-stimulated monocyte cultures. Cultures indicated by “Control”contained PBMCs cultured for five days in the presence of IL-4 andGM-CSF, then for a further 48 hours in TNF-α. Cultures indicated as“with CsA” contained PBMCs cultured for five days in the presence ofIL-4 and GM-CSF, then for a further 48 hours in TNF-α+1 ug/mlcyclosporine A. Cultures indicated as “MSC” contained PBMCs cultured forfive days in the presence of IL-4 and GM-CSF, then for a further 48hours in TNF−α+10⁴ MSCs. Cultures indicated as “SB623” contained PBMCscultured for five days in the presence of IL-4 and GM-CSF, then for afurther 48 hours in TNF−α+10⁴ SB623 cells. The results for MSCs andSB623 cells are the average of three experiments, each using a samplefrom a different donor. Monocyte donor was the same in all cases. Allcultures were started with 10⁵ PBMCs.

FIGS. 20A and 20B show measurements of cytokine expression in monocytes.FIG. 20A shows the percentage of monocytes in culture that express theinflammatory cytokine TNF-α. FIG. 20B shows the percentage of monocytesin culture that express the anti-inflammatory cytokine IL-10. Monocytes,selected on the basis of surface expression of CD 14, were culturedwithout supplement (“negative”), with macrophage colony-stimulatingfactor (“MCSF”), with granulocyte/macrophage colony-stimulating factor(“GMCSF”), with MSCs or with SB623 cells. MSCs and SB623 cells wereobtained from three different donors, indicated as D52, D55 and D65 inthe figure.

DETAILED DESCRIPTION

Practice of the present disclosure employs, unless otherwise indicated,standard methods and conventional techniques in the fields of cellbiology, toxicology, molecular biology, biochemistry, cell culture,immunology, oncology, recombinant DNA and related fields as are withinthe skill of the art. Such techniques are described in the literatureand thereby available to those of skill in the art. See, for example,Alberts, B. et al., “Molecular Biology of the Cell,” 5^(th) edition,Garland Science, New York, N.Y., 2008; Voet, D. et al. “Fmundamentals ofBiochemistry: Life at the Molecular Level,” 3^(rd) edition, John Wiley &Sons, Hoboken, N.J., 2008; Sambrook, J. et al., “Molecular Cloning: ALaboratory Manual,” 3^(rd) edition, Cold Spring Harbor Laboratory Press,2001; Ausubel, F. et al., “Current Protocols in Molecular Biology,” JohnWiley & Sons, New York, 1987 and periodic updates; Freshney, R. I.,“Culture of Animal Cells: A Manual of Basic Technique,” Fifth Edition,Wiley, New York. 2005; and the series “Methods in Enzymology,” AcademicPress, San Diego, Calif. Standard techniques in immunology aredescribed, for example, in “Current Protocols in Immunology,” (R. Coico,series editor), Wiley, updated August 2010.

For the purposes of the present disclosure, the term “peripheral” isused to refer to portions of the body outside of the central nervoussystem. These include, for example, the bone marrow, peripheralcirculation and lymphoid organs.

Preparation of SB623 Cells

Mesenchymal stem cells (MSCs) can be obtained by selecting adherentcells from bone marrow, and can be induced to form SB623 cells byexpression of the Notch intracellular domain (NICD) in the adherentcells. In one embodiment, a culture of MSCs is contacted with apolynucleotide comprising sequences encoding a NICD (e.g., bytransfection), followed by enrichment of transfected cells by drugselection and further culture. See, for example, U.S. Pat. No. 7,682,825(Mar. 23, 2010); U.S. Patent Application Publication No. 2010/0266554(Oct. 21, 2010); and WO 2009/023251 (Feb. 19, 2009); all of whichdisclosures are incorporated by reference, in their entireties, for thepurposes of describing isolation of mesenchymal stem cells andconversion of mesenchymal stem cells to SB623 cells (denoted “neuralprecursor cells” and “neural regenerating cells” in those documents).See also Example 1, infra.

In these methods, any polynucleotide encoding a Notch intracellulardomain (e.g., vector) can be used, and any method for the selection andenrichment of transfected cells can be used. For example, in certainembodiments, a vector containing sequences encoding a Notchintracellular domain also contains sequences encoding a drug resistancemarker (e.g. resistance to G418). In these embodiments, selection isachieved, after transfection of a cell culture with the vector, byadding a selective agent (e.g., G418) to the cell culture in an amountsufficient to kill cells that do not comprise the vector but spare cellsthat do. Absence of selection entails removal of said selective agent orreduction of its concentration to a level that does not kill cells thatdo not comprise the vector.

Senescence in SB623 Cells

As described above, SB623 cells are derived from MSCs by expression of aNICD in cultured MSCs. Because MSCs that have undergone manipulation inculture often become senescent; the SB623 cells derived therefrom weretested for senescence.

SB623 cells do not form colonies in soft agar, indicating that they arenot transformed cells. In addition, when SB623 cells were prelabelledwith carboxyfluorescein diacetate succinimidyl ester (CFSE), acell-permeable dye that is diluted by cell division, a sub-population ofcells retained high concentrations of CFSE after 5 days of culture (FIG.1). This slowly-proliferating (or non-proliferating) sub-population wasnot observed in MSC cultures. Certain cells in the SB623 cell populationwere also observed to stain intensely for beta-galactosidase (a markerof cell senescence) and such cells were more plentiful in SB623 culturesthan in MSC cultures. These results are consistent with the existence ofa pool of non-proliferating, senescent cells in the SB623 cellpopulation.

Cell proliferation was measured by plating one million MSCs or SB623cells and, after three days in culture, measuring viable cells by TrypanBlue exclusion. FIG. 2 shows that a higher number of viable cells wereobserved in the MSC cultures, indicating a lower proliferative index forthe SB623 cells. Cell cycle status was assessed by propidium iodidestaining, which revealed a higher proportion of cells in resting phase(G0/G1) in SB623 cultures (FIGS. 3A and 3B), providing further supportfor a reduced rate of proliferation in SB623 cells.

An additional assessment of senescence was conducted by stainingpopulations of SB623 cells for expression of the p16Ink4A protein.p16Ink4A inhibits the progression from the G1 to S phases of the cellcycle and is expressed in senescent cells. FIG. 4 shows that a higherpercentage of p16Ink4A-expressing cells were detected in cultures ofSB623 cells, compared to MSCs. Moreover, when cells in SB623 culturesthat retained high CFSE levels after 5 days of culture were tested forp16Ink4A expression, the sub-population of SB623 cells expressingp16Ink4A coincided with the fraction containing high CFSE levels. Theseresults, taken together, indicate the existence of a subpopulation ofsenescent cells within SB623 cultures.

Surface Marker and Cytokine Expression

SB623 cells express a number of surface markers in common with MSCs.These include CD29, CD44, CD73, CD90, CD105 and vascular cell adhesionmolecule-1 (VCAM-1 or CD 106). Levels of CD44 and CD73 were higher, andVCAM-1 levels were lower, in SB623 cells compared to MSCs. SB623 cellsalso express intercellular adhesion molecule-1 (ICAM-1 or CD54), whichis not normally expressed by MSCs. See FIGS. 5 and 6. MSCs and SB623cells do not express the surface markers CD31, CD34 and CD45.

SB623 cells also secrete a number of cytokines and trophic factors. Theidentity of certain of these factors was determined by blocking proteinsecretion with Brefeldin A and testing for intracellular cytokines byantibody staining and flow cytometry. These studies showed that SB623cells produce, among other factors, interleukin 1α (IL-1α),interleukin-6 (IL-6), granulocyte/macrophage colony-stimulating factor(GM-CSF), vascular endothelial growth factor-A (VEGF-A) and transforminggrowth factor beta-1 (TGF β−1). See FIGS. 7 and 8. Amounts of IL-6 andGM-CSF produced by SB623 cells were generally greater than thoseproduced by MSCs.

Because senescent cells have been reported to synthesize and secretecertain growth-stimulatory cytokines and trophic factors (Orjalo et al.(2009) Proc. Natl. Acad. Sci. USA 106:17031-17036), the existence of apopulation of senescent cells within SB623 cultures suggested theutility of SB623 cell transplantation to support various types of tissueregeneration. However, the secretory profile of senescent cells has alsobeen reported to be pro-inflammatory, which, if it were the case forSB623 cells, might reduce the usefulness of SB623 cells for celltransplantation therapy.

Surprisingly, and despite the presence of a population of senescentcells in SB623 cultures, SB623 cells possess a number ofimmunosuppressive properties, as disclosed herein. For example, SB623cells suppress proliferation and activation of T-cells, alter thecytokine profile of T-cells, block the differentiation of monocytes todendritic cells, and are superior to their parental MSCs at slowingmaturation of dendritic cells.

Suppression of T-Cell Activation and T-Cell Proliferation by SB623 Cells

SB623 cells were added to mixed lymphocyte reactions (MLRs) containing10⁵ CFSE-labeled peripheral blood T-cells and 10⁵ peripheral bloodmononuclear cells from an unrelated donor. Levels of CD69, an earlymarker of T-cell activation, were measured to examine the ability ofSB623 cells to modulate T-cell activation. In control mixed lymphocytereactions, surface expression of CD69 was robustly induced. However,after one day in the presence of 10⁴ SB623 cells, the fraction of CD4⁺T-cells (i.e., helper T-cells) in the MLR expressing surface CD69 wassignificantly reduced. See Example 4.

After five days in the presence of SB623 cells, dilution of CFSE inprelabelled CD4⁺ T-cells (indicative of cell proliferation) indicatedthat proliferation of CD4⁺ T-cells in the MLR was reduced in thepresence of SB623 cells. See Example 4. Thus, SB623 cells are capable ofsuppressing both T-cell proliferation and T-cell activation.

Additional effects of SB623 cells on T-cell function included reductionof surface HLA-DR expression (Example 4 herein), increased production ofregulatory T-cells in in vitro cultures of naive T-cells (Example 6herein) and alteration of cytokine secretion (Examples 7 and 8 herein).SB623 cells were also effective at reducing T-cell proliferation in axenogenic lymphocyte activation system. See Example 5 herein.

Inhibition of Dendritic Cell Development by SB623 Cells

Differentiation of monocytes into dendritic cells (a type ofantigen-presenting cell) and further maturation of dendritic cells canbe stimulated in vitro by exposure of monocytes to the cytokinesinterleukin-4 (IL-4) and granulocyte/macrophage colony-stimulatingfactor (GM-CSF). This differentiation can be blocked by interleukin-6(IL-6) or vascular endothelial growth factor (VEGF), both of which areamong the cytokines known to be secreted by SB623 cells. See, forexample. Tate et al. (2010) Cell Transplant. 19:973-984 and WO2009/023251.

The inventors show herein that co-culture of monocytes with SB623 cellsreduces both the differentiation of monocytes into CD 1a⁺ dendriticcells and the maturation of dendritic cells to a CD86⁺ status. SeeExamples 9 and 10 infra. Because of their abilities to reduce productionof new dendritic cells and inhibit the function of existing dendriticcells, SB 623 cells can be used to treat and/or ameliorategraft-versus-host-disease (GVHD) resulting from activation of T-cells bypresentation of peptides by antigen-presenting cells, such as dendriticcells.

Because of their various immunosuppressive properties as describedherein, SB623 cells can be used in place of other biological andchemical immunosuppressants (e.g., cyclosporine, tacrolimus, sirolimus,interferons, mycophenolic acid, fingolimod, myriocin, azathioprine,mercaptopurine, dactinomycin, mitomnycin C, bleomycin, mithramycin,anthracyclines, methotrexate, FK506, cyclophosphamides, nitrosoureas,platinum compounds and glucocorticoids). Moreover, use ofimmunosuppressive agents is not required to accompany SB623 allogeneictransplantation in cell therapy, e.g., for neuroregeneration andtreatment of nervous system disorders.

Progenitor Cells

Progenitor cells, which can be converted to SB623 cells, can be any typeof non-terminally differentiated cell. For example, totipotent stemcells as disclosed for example, in U.S. Pat. Nos. 5,843,780; 6,200,806and 7,029,913 can be used as progenitor cells. Totipotent stem cells canbe cultured (e.g., U.S. Pat. Nos. 6,602,711 and 7,005,252) anddifferentiated into various types of pluripotent cells (e.g., U.S. Pat.Nos. 6,280,718; 6,613,568 and 6,887,706), which can also be used asprogenitor cells in the practice of the disclosed methods.

Another exemplary type of progenitor cells are marrow adherent stromalcells (MASCs), also known as marrow adherent stem cells, bone marrowstromal cells (BMSCs) and mesenchymal stem cells (MSCs). Exemplarydisclosures of MASCs are provided in U.S. patent application publicationNo. 2003/0003090; Prockop (1997) Science 276:71-74 and Jiang (2002)Nature 418:41-49. Methods for the isolation and purification of MASCscan be found, for example, in U.S. Pat. No. 5,486,359; Pittenger et al.(1999) Science 284:143-147 and Dezawa et al. (2001) Eur. J. Neurosci.14:1771-1776. Human MASCs are commercially available (e.g.,BioWhittaker, Walkersville, Md.) or can be obtained from donors by,e.g., bone marrow aspiration, followed by selection for adherent bonemarrow cells. See, e.g., WO 2005/100552.

MASCs can also be isolated from umbilical cord blood. See, for example,Campagnoli et al. (2001) Blood 98:2396-2402; Erices et al. (2000) Br. J.Haematol. 109:235-242 and Hon et al. (2003) Int. J. Hematol. 78:256-261.

Conversion of MSCs to SB623 cells has been described, for example, inU.S. Pat. No. 7,682,825 (Mar. 23, 2010) and WO 2009/023251 (Feb. 19,2009); both of which disclosures are incorporated by reference, in theirentireties, for the purposes of describing isolation of mesenchymal stemcells and conversion of mesenchymnal stem cells to SB623 cells (denoted“neural precursor cells” and “neural regenerating cells” in thosedocuments).

Notch Intracellular Domain

The Notch protein is a transmembrane receptor, found in all metazoans,that influences cell differentiation through intracellular signaling.Contact of the Notch extracellular domain with a Notch ligand (e.g.,Delta, Serrate, Jagged) results in two proteolytic cleavages of theNotch protein, the second of which is catalyzed by a γ-secretase andreleases the Notch intracellular domain (NICD) into the cytoplasm. Inthe mouse Notch protein, this cleavage occurs between amino acidsgly1743 and val1744. The NICD translocates to the nucleus, where it actsas a transcription factor, recruiting additional transcriptionalregulatory proteins (e.g., MAM, histone acetylases) to relievetranscriptional repression of various target genes (e.g., Hes 1).

Additional details and information regarding Notch signaling are found,for example in Artavanis-Tsakonas et al. (1995) Science 268:225-232;Mumm and Kopan (2000) Develop. Biol. 228:151-165 and Ehebauer et al.(2006) Sci. STKE 2006 (364), cm7. [DOI: 10.1126/stke.3642006cm7].

Cell Culture and Transfection

Standard methods for cell culture are known in the art. See, forexample, R. I. Freshney “Culture of Animal Cells: A Manual of BasicTechnique,” Fifth Edition, Wiley, New York, 2005.

Methods for introduction of exogenous DNA into cells (i.e.,transfection) are also well-known in the art. See, for example, Sambrooket al. “Molecular Cloning: A Laboratory Manual,” Third Edition, ColdSpring Harbor Laboratory Press, 2001; Ausubel et al., “Current Protocolsin Molecular Biology,” John Wiley & Sons, New York, 1987 and periodicupdates.

Autoimmune Disorders and Allergic Reactions

Autoimmune disorders result from an immune response that attacks normalhealthy tissue. Exemplary autoimmune disorders include, but are notlimited to, amyotrophic lateral sclerosis, ankylosing spondylitis,thrombocytopenic purpura, Hashimoto's thyroiditis, Guillain Barresyndrome, pernicious anemnia, dermatosyositis, Addison's disease, Type Idiabetes, rheumatoid arthritis, systemic lupus erythematosus (“lupus”),dermatomyositis, Sjogren's syndrome, multiple sclerosis, Myastheniagravis, polymyositis, biliary cirrhosis, psoriasis, reactive arthritis,Grave's disease, ulcerative colitis, inflammatory bowel disease,vasculitis, Crohn's disease, and celiac disease-sprue (gluten sensitiveenteropathy).

Allergies result from an immune hypersensitivity to external substancesthat would not normally stimulate an immune response. Common allergensinclude pollen, mold, pet dander and dust. Certain foods and drugs canalso cause allergic reactions.

The immunosuppressive properties of SB623 cells, as disclosed herein,make SB623 cells useful for the treatment of autoimmune disorders andallergies.

Formulations, Kits and Routes of Administration

Therapeutic compositions comprising SB623 cells as disclosed herein arealso provided. Such compositions typically comprise the cells and apharmaceutically acceptable carrier.

The therapeutic compositions disclosed herein are useful for, interalia, immunomodulation (e.g., reducing immune activation) and reversingthe progression of various immune disorders. Accordingly, a“therapeutically effective amount” of a composition comprising SB623cells can be an amount that prevents or reverses immune activation. Forexample, dosage amounts can vary from about 100; 500; 1,000; 2,500;5,000; 10,000; 20,000; 50,000; 100,000; 500,000; 1,000,000; 5,000,000 to10,000,000 cells or more; with a frequency of administration of, e.g.,once per day, twice per week, once per week, twice per month, once permonth, depending upon, e.g., body weight, route of administration,severity of disease, etc.

Supplementary active compounds can also be incorporated into thecompositions. For example, SB623 cells are useful in combination withother immune modulators such as cyclosporine for treatment of, e.g.,autoimmune disease or to inhibit transplant rejection and/or GVHD.Accordingly, therapeutic compositions as disclosed herein can containboth SB623 cells and cyclosporine (or any other immunosuppressant). Whena composition of SB623 cells is used in combination with anothertherapeutic agent, one can also refer to the therapeutically effectivedose of the combination, which is the combined amounts of the SB623cells and the other agent that result in immunomodulation, whetheradministered in combination, serially or simultaneously. More than onecombination of concentrations can be therapeutically effective.

Various pharmaceutical compositions and techniques for their preparationand use are known to those of skill in the art in light of the presentdisclosure. For a detailed listing of suitable pharmacologicalcompositions and techniques for their administration one may refer totexts such as Remington's Pharmaceutical Sciences, 17th ed. 1985;Brunton et al., “Goodman and Gilman's The Pharmacological Basis ofTherapeutics,” McGraw-Hill, 2005; University of the Sciences inPhiladelphia (eds.). “Remington: The Science and Practice of Pharmacy,”Lippincott Williams & Wilkins, 2005; and University of the Sciences inPhiladelphia (eds.), “Remington: The Principles of Pharmacy Practice,”Lippincott Williams & Wilkins, 2008.

The cells described herein may be suspended in a physiologicallycompatible carrier for transplantation. As used herein, the term“physiologically compatible carrier” refers to a carrier that iscompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof. Those of skill in the art arefamiliar with physiologically compatible carriers. Examples of suitablecarriers include cell culture medium (e.g., Eagle's minimal essentialmedium), phosphate buffered saline, Hank's balanced saltsolution+/−glucose (HBSS), and multiple electrolyte solutions such asPlasma-Lyte™ A (Baxter).

The volume of a SB623 cell suspension administered to a patient willvary depending on the site of implantation, treatment goal and number ofcells in solution. Typically the amount of cells administered to apatient will be a therapeutically effective amount. As used herein, a“therapeutically effective amount” or “effective amount” refers to thenumber of transplanted cells which are required to effect treatment ofthe particular disorder; i.e., to produce a reduction in the amountand/or severity of the symptoms associated with that disorder. Atherapeutically effective amount further refers to that amount of thecomposition sufficient to result in full or partial amelioration ofsymptoms of the relevant medical condition, or an increase in rate oftreatment, healing, prevention or amelioration of such condition. Forexample, in the case of treatment for graft-versus-host disease,transplantation of a therapeutically effective amount of SB623 cellstypically results in immunosuppression of grafted cells. If the disorderis graft rejection, for example, a therapeutically effective amount isthat number of SB623 which, when transplanted, results in sufficientimmunosuppression in the host such that a graft is accepted.Therapeutically effective amounts will vary with the type of disease ordisorder, extensiveness of the disease or disorder, and size of theorganism suffering from the disease or disorder.

The disclosed therapeutic compositions further include pharmaceuticallyacceptable materials, compositions or vehicle, such as a liquid or solidfiller, diluent, excipient, solvent or encapsulating material, i.e.,carriers. These carriers can, for example, stabilize the SB623 cellsand/or facilitate the survival of the SB623 cells in the body. Eachcarrier should be “acceptable” in the sense of being compatible with theother ingredients of the formulation and not injurious to the subject.Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations. Wetting agents, emulsifiers andlubricants, such as sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, release agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the compositions.

Another aspect of the present disclosure relates to kits for carryingout the administration of SB623 cells, optionally in combination withanother therapeutic agent, to a subject. In one embodiment, a kitcomprises a composition of SB623 cells, formulated in a pharmaceuticalcarrier, optionally containing, e.g., cyclosporine or anotherimmunosuppressant, formulated as appropriate, in one or more separatepharmaceutical preparations.

Exemplary formulations include, but are not limited to, those suitablefor parenteral administration, e.g., intrapulmonary, intravenous,intra-arterial, intra-ocular, intra-cranial, sub-meningial, orsubcutaneous administration, including formulations encapsulated inmicelles, liposomes or drug-release capsules (active agents incorporatedwithin a biocompatible coating designed for slow-release); ingestibleformulations; formulations for topical use, such as eye drops, creams,ointments and gels; and other formulations such as inhalants, aerosolsand sprays. The dosage of the compositions of the disclosure will varyaccording to the extent and severity of the need for treatment, theactivity of the administered composition, the general health of thesubject, and other considerations well known to the skilled artisan.

In additional embodiments, the compositions described herein aredelivered locally. Localized delivery allows for the delivery of thecomposition non-systemically, thereby reducing the body burden of thecomposition as compared to systemic delivery. Such local delivery can beachieved, for example, through the use of various medically implanteddevices including, but not limited to, stents and catheters, or can beachieved by inhalation, phlebotomy, injection or surgery. Methods forcoating, implanting, embedding, and otherwise attaching desired agentsto medical devices such as stents and catheters are established in theart and contemplated herein.

EXAMPLES Example 1 Preparation of MSCs and SB623 Cells

Bone marrow aspirates from adult human donors were obtained from LonzaWalkersville, Inc. (Walkersville. Md.) and plated in α-MEM (Mediatech,Herndon, Va.) supplemented with 10% fetal bovine serum (Hyclone, Logan,Utah), 2 mM L-glutamine (Invitrogen, Carlsbad, Calif.) andpenicillin/streptomycin (Invitrogen). Cells were cultured for three daysat 37.degree. C. and 5% CO₂, to obtain a monolayer of adherent cells.After removal of non-adherent cells, culture was continued under thesame conditions for two weeks. During this time, cells were passagedtwice, using 0.25% trypsin/EDTA. A portion of the cells from the secondpassage were frozen as MSCs.

The remaining cells from the second passage were plated and transfected,using Fugene6 (Roche Diagnostics, Indianapolis, Ind.), with a plasmidcontaining sequences encoding a Notch intracellular domain operativelylinked to a cytomegalovirus promoter (pCMV-hNICD1-SV40-Neo^(R)). Thisplasmid also contained sequences encoding resistance to neomycin andG418 under the transcriptional control of a SV40 promoter. Transfectedcells were cultured at 37.degree. C. and 5% CO₂ in the growth mediumdescribed in the previous paragraph, supplemented with 100 .mu.g/ml G418(Invitrogen, Carlsbad, Calif.). After seven days, G418-resistantcolonies were expanded and the culture was passaged twice. After thesecond passage, the cells were collected and frozen as SB623 cells.

MSCs and SB623 cells prepared as described herein were thawed asrequired and used for further study.

Example 2 Proliferative Capacity of MSCs and SB623 Cells

To quantify cell proliferation, one million MSCs or SB623 cells wereplated and cultured for three days. Viable cells were counted by trypanblue exclusion on Day 3. FIG. 2 shows that fewer live cells were presentin the SB623 cultures, compared to the MSC cultures.

The cell cycle profile of MSC and SB623 cultures was assessed bypropidium iodide staining. Propidium iodide is a DNA-intercalating dyethat stains cells in the resting phase of the cell cycle more stronglythan proliferating cells. After three days of culture, one million MSCsor SB623 cells were fixed in 70% ethanol overnight at 4.degree. C. Aftertwo washes in PBS/2% FBS, cells were incubated in one ml of stainingbuffer (50 .mu.g/ml propidium iodide, 50 .mu.g/ml RNAse) (Sigma, St.Louis, Mo.) in PBS/2% FBS for 30 min in the dark. Acquisition andanalysis were done on a FACSCAIibur™ flow cytometer (BD Biosciences)using a CellQuestPro™ program (BD Biosciences, San Jose, Calif.) on theFL-2 linear channel. FIGS. 3A and 3B show greater propidium iodidestaining of SB623 cells, compared to MSCs, indicating a higher fractionof cells in the G0/G1 resting phase of the cell cycle in SB623 cellcultures.

Dilution of the cell-autonomous dye 5-(-6-)carboxyfluorescein diacetate(CFSE) was used as an additional measure of the kinetics ofproliferation. For this analysis, an equal number of MSCs and SB623cells were labeled for 2 min at room temperature with of 5 .mu.M of5-(-6-)carboxyfluorescein diacetate (Invitrogen, Carlsbad, Calif.), thencultured for five days. Flow cytometry acquisition and analysis (forCFSE) were done on a FACSCAlibur™ flow cytometer (BD Biosciences) usingthe FL-1 log channel. The results, (FIG. 1) show that SB623 cellcultures contained a population of cells with high CFSE content,compared to MSCs, indicating the presence, in SB623 cell cultures, of apopulation of non-dividing or slowly-dividing cells.

The levels of intracellular p16Ink4A protein in MSCs and SB623 cellswere assessed as follows. Cells were cultured for three days, then fixedwith 4% paraformaldehyde and permeabilized with PBS containing 0.1%Triton X-100. After two washes in PBS containing 2% fetal bovine serum(PBS/2% FBS), cell pellets were resuspended in 0.2 ml of PBS/2% FBS anddivided into two samples. One cell sample was stained with phycoerythrin(PE)-conjugated anti-p16Ink4A antibody (BD Biosciences, San Jose,Calif.) and the other sample was incubated with PE-conjugated mouse IgGas an isotype control. Samples were analyzed by flow cytometry on aFACSCAlibur™ flow cytometer (BD Biosciences) and the data was convertedto percentage of cells in the culture expressing p16Ink4A by gating oncells that stained positive for p16Ink4A and negative for IgG. FIG. 4shows that SB623 cell cultures contain a significantly higher fractionof cells expressing p16Ink4A.

Example 3 Surface Marker and Cytokine Expression by MSCs and SB623 Cells

For measurements of cell surface markers. MSCs or SB623 cells wereharvested from culture using 0.25% Trypsin/EDTA (Invitrogen, Carlsbad,Calif.), washed in PBS/2% FBS and resuspended in 1 ml of PBS/2% FBS.Cells were incubated with fluorochrome conjugated antibody to CD29,CD31, CD34, CD44, CD45, CD73, CD90 (all from BD Biosciences, San Jose,Calif.) or CD105 (Invitrogen, Carlsbad, Calif.) for 15 min on ice. Cellswere then washed once with PBS/2% FBS and acquired on a FACSCalibur™flow cytometer (BD Biosciences, San Jose, Calif.). The CellQuestPro™software (BD Biosciences) was used for data analysis. Results wereexpressed as dMFI (“delta mean fluorescence intensity”), using IgG as acontrol; i.e., MFI for IgG was subtracted from the MFI obtained for agiven surface marker to obtain the dMFI.

The results are shown in FIGS. 5 and 6. FIG. 5 shows that, although bothMSCs and SB623 cells express CD44, CD73 and CD105, SB623 cellsconsistently express higher levels of these surface markers. FIG. 6shows that SB623 cells also express consistently higher levels of CD54than do MSCs.

For detection of intracellular cytokines, cells were cultured for threedays and treated with a 1:1,000 dilution of Brefeldin A (eBioscience,San Diego, Calif., final concentration of 3 ug/ml) for six hours priorto harvest. Cells were fixed and permeabilized as described above formeasurement of intracellular pInk4A, and incubated withfluorochrome-conjugated antibodies to human GM-CSF (BD), IL-1 alpha(eBioscience, San Diego, Calif.), IL-6 (BD) or TGF β-1 (R&D Systems,Minneapolis, Minn.) for one hour followed by two washes with PBS/2% FBS.Data acquisition and analysis was performed on a BD FACSCalibur™instrument using CellQuestPro™ software.

The results of these analyses, presented in FIG. 7 show roughlyequivalent levels of expression of IL-1α, IL-6 and GM-CSF by MSCs andSB623 cells; while FIG. 8 shows that comparable levels of TGF-β-1 andVEGF-A are produced by MSCs and SB623 cells.

Example 4 Allogeneic Mixed Lymphocyte Reaction (Allo-MLR)

Cells for allogeneic mixed lymphocyte reactions were obtained from 10 mlsamples of peripheral blood from healthy, unrelated individuals. Toobtain responder T-cells, a RosetteSep T-cell enrichment kit (StemcellTechnologies, Vancouver, BC, Canada) was used according to themanufacturer's specifications. Enriched T-cells (responder cells) werelabeled for 2 minutes at room temperature with 5 uM5-(-6-)carboxyfluorescein diacetate (CFSE), obtained from Invitrogen,Carlsbad, Calif. After serum quenching and three washes in PBS, thelabeled responder cells were plated, in a volume of 0.1 ml of completelymphocyte medium (RPMI (Mediatech, Manassas, Va.)+10% FBS (Lonza,Allendale. N.J.) containing 10⁵ cells, in the well of a 96-well U-bottomplate.

To prepare stimulator cells, peripheral blood buffy, coat mononuclearcells were recovered after Ficoll™ density gradient centrifugation. Redcell lysis buffer (Sigma-Aldrich, St. Louis, Mo.) was added for 10 minat 37.degree. C.; then the cells were washed twice with PBS/2%heat-inactivated FBS. The mononuclear stimulator cells were either addedto the well containing responder cells (10⁵ cells in a volume of 0.1 ml)or 10⁵ stimulator cells were mixed with 10⁴ SB623 cells or 10⁴ MSCs,centrifuged and the pelleted cells resuspended in a volume of 0.1 ml ofcomplete lymphocyte medium (as described above) which was then added toa well of CFSE-labeled responder cells prepared as described above.

Display of CD69 (an early T-cell activation marker) on the surface ofCD4⁺ T-cells in the culture, two days after initiation of the reaction,was used as an assay for T-cell activation. For analysis of CD69expression, cells were harvested by pipette after two days, stained witha peridinin chlorophyll protein (PerCP)-conjugated anti-CD69 antibody(eBioscience, San Diego, Calif.), and analyzed using a FACSCalibur™ flowcytometer (Becton, Dickinson & Co., San Jose, Calif.), gating on CD4⁺lymphocytes.

For measurements of T-cell proliferation, cells were harvested afterseven days of culture and stained with a phycoerythrin (PE)-conjugatedanti-CD4 antibody (BD). A BD FACSCalibur flow cytometer was used fordata acquisition.

In a control allo-MLR, the fraction of T-cells within the CD4⁺population, in which expression of surface CD69 had been induced, wassignificantly increased after two days (FIGS. 9A and 9B).

The effect of co-culture with MSCs and SB623 cells on T-cell activationin the MLR was also assessed. In these experiments, 10,000 MSCs or10,000 SB623 cells were added to the culture at the start of the MLR.Under these conditions, the increase in surface CD69-expressing cellsthat was observed in control cultures after two days was significantlyreduced by co-incubation with MSCs or SB623 cells (p<0.05; FIGS. 9A and9B).

Another measure of T-cell activation, the proliferation rate of CD⁺T-cells was assayed 7 days after initiation of the MLR. For theseexperiments, cells were harvested from the MLR by pipette and stainedwith a PE-labeled anti-CD4 antibody. Flow cytometry was conducted usinga Becton-Dickinson FACSCalibur™ apparatus, gating on CD4⁺ cells; anddilution of CSFE was evaluated as an indicator of the proliferation rateof the CD4⁺ responder T-cells. In a control allo-MLR, more than 80% ofthe CD4⁺ responder T-cells had proliferated after seven days. In thepresence of SB623 cells or MSCs, T-cell proliferation was significantlyreduced (i.e., higher levels of CFSE staining were observed, FIGS. 10Aand 10B).

Induction of surface HLA-DR expression is also a measure of T-cellactivation. Both SB623 cells and MSCs reduced the percentage ofHLA-DR-expressing T-cells in the allo-MLR (FIG. 11).

Thus, by a number of different, independent criteria, SB623 cellssuppressed T-cell activation. The ability to block T-cell activationindicates the usefulness of SB623 cells for immunosuppression.

Example 5 Xenogeneic Lymphocyte Activation Reaction

The immunosuppressive properties of SB623 cells were also demonstratedin a xenogenic transplantation model system. Xenogenic lymphocytereactions were established using Sprague-Dawley rat glial mix cells(comprising astrocytes and microglial cells) as stimulators and humanperipheral blood T-cells, labeled with PKH26 according to themanufacturer's instructions (Sigma-Aldrich, St. Louis, Mo.), asresponders. To obtain glial mix cells, postnatal day 9 rat brains wereharvested and triturated prior to treatment with 0.25% Trypsin for 30min Cell suspensions were filtered through a 70 .nm.M cell strainer andoverlaid on Ficoll™ prior to centrifugation. Glial mix cells werecultured in DMEM/F12/10% FBS/pen-strep for 14 days prior to use in theassay. The xenogeneic reaction was performed using cell ratios similarto those used in the allogeneic MLR (100,000 glial mix cells: 100,000CFSE-labeled human T-cells; and optionally 10,000 MSCs or SB623 cells)over a 5-day period. PKH26 dilution in human CD3-gated T-cells (whichincludes both CD4⁺ and CD8⁺ T-cells) was assessed by flow cytometry.

As in the allogeneic MLR, addition of SB623 cells or MSCs to thexenogeneic system reduced the degree of proliferation of responderT-cells otherwise observed after stimulation by the glial mix cells(FIG. 12). Thus, the immunosuppressive properties of MSCs and SB623cells are not limited to autologous or allogeneic environments.

Example 6 Effect of SB623 Cells on Development of Regulatory T-Cells

Regulatory T-cells (T_(reg)s) are capable of dampening or suppressingimmune responses. Accordingly, the ability of SB623 cells to support thegeneration of T_(reg)s was investigated. To this end, enriched T-cellsfrom peripheral blood, purified as described in Example 2, were culturedin the presence of interleukin-2 (IL-2), which has been shown tostimulate the differentiation of naive T-cells into T_(reg)s, and theeffect of co-culture with MSCs or SB623 cells on this process wasassessed. Co-cultures contained a 10:1 ratio of T-cells to SB623 cellsor a 10:1 ratio of T-cells to MSCs (10⁵ T-cells:10⁴ MSCs or SB623cells). Coexpression of the surface markers CD4 and CD25, secretion ofthe cytokine interleukin-10 (IL-10) and intracellular production of thetranscription factor FoxP3 were used as markers for T_(reg)s.

For these experiments, human T-cells were enriched from peripheral bloodusing a T-cell isolation kit (StemCell Technologies, Vancouver, Canada)according to the manufacturer's protocol. Enriched T cells were culturedovernight in RPMI-1640/10% heat-inactivated FBS/pen/strep prior to use.On Day-1, 10,000 MSCs or SB623 cells were plated per well in 96-wellU-bottom plates. On Day 0 of the co-culture assay, 100,000 enriched Tcells were transferred to each well of pre-established MSC or SB623 cellmonolayer also containing 10 ng/ml IL-2. As internal controls, T-cellcultures were also maintained in the absence of MSCs or SB623 cells. Onday 7, cells were stained for surface CD4 (a helper T-cell marker) andCD25 (the IL-2 receptor alpha chain), and for intracellular FoxP3.

The results of the assays for the surface markers CD4 and CD25 are shownin FIGS. 13A and 13B. Co-culture of SB623 cells with IL-2-stimulatedT-cells significantly increased the number of CD⁺CD25⁺ T_(reg) cells(compare left-most and right-most panels of FIG. 13A) and that thisstimulation of T_(reg) development was greater when the T-cells wereco-cultured with SB623 cells than when they were co-cultured with MSCs(compare center and right panels of FIG. 13A).

Assays for the forkhead box P3 (FoxP3) protein confirmed these results.FoxP3 is a transcription factor that regulates the development andfunction of T_(reg)s. Intracellular FoxP3 was detected by culturing orco-culturing T-cells for seven days (as described above), then fixingand permeabilizing cells with CytoFix/Perm (eBioscience, San Diego,Calif.). PE-conjugated anti-FoxP3 antibody (clone PCH101, eBioscience,1:50 dilution) was used to stain cells for 30 min, and stained cellswere analyzed by flow cytometry gating on lymphocytes based on cellsize. The results, shown in FIG. 14A and 14B, demonstrate thatco-culture with MSCs and SB623 cells increased FoxP3 expression byT-cells, in the presence of IL-2, compared to its expression in T-cellsthat were not co-cultured.

One mechanism of immunosuppression by T_(reg)s is through secretion ofanti-inflammatory cytokines such as, for example, interleukin-10(IL-10). Accordingly, the percentage of T-cells producing IL-10 inIL-2-containing T-cell cultures, or in co-cultures with MSCs or SB623s,was determined by staining for intracellular IL-10 with afluorochrome-conjugated anti-IL-10 antibody after seven days of cultureor co-culture.

Accordingly, after 7 days of culture or co-culture, cells were treatedwith a 1:1,000 dilution of Brefeldin A (eBioscience, San Diego Calif.)(to prevent secretion of extracellular proteins) for six hours, fixedwith 2% paraformaldehyde for 15 min, then permeabilized with 0.05% (v/v)Triton-X-100 in PBS/2% FBS for 15 min on ice. Alexa 488-conjugatedanti-human IL-10 antibody (eBioscience, San Diego, Calif.) was thenadded and the cultures were incubated on ice for 30 min Wells werewashed twice with 2% fetal bovine serum/0.01% (v/v) Tween 20; cells wereacquired by pipette and analyzed using a FACSCalibur™ flow cytometer(Becton, Dickinson & Co., San Jose, Calif.). Data analysis was conductedusing CellQuestPro™ software (Becton, Dickinson & Co., San Jose,Calif.).

Results of this analysis revealed that T-cells cultured in the presenceof IL-2 did not express intracellular IL-10; while low levels of IL-10were produced by CD4⁺ T-cells when they were co-cultured with eitherSB623 cells or MSCs in the presence of IL-2, with slightly more IL-10being produced by T-cells that were co-cultured with SB623 cells (FIGS.15A and 15B).

Example 7 Conversion of Pro-Inflammatory to Anti-Inflammatory CytokineProfile by SB623 Cells

The effect of co-culture of MSCs and SB623 cells, on the relativeamounts of pro- and anti-inflammatory cytokines produced by T-cells, wasassessed by measuring levels of IL-10 (an anti-inflammatory cytokine)and interferon-gamma (IFN-γ, a pro-inflammatory cytokine) in T-cellsthat had been sub-optimally activated by treatment with phorbolmyristate acetate (PMA) and ionomycin. For these experiments, T-cellswere enriched from peripheral blood and cultured, or co-cultured withMSCs or SB623 cells, as described above (Example 6), except that culturewas conducted in the absence of IL-2. On Day 7, non-activating doses of25 ng/ml of phorbol 12-myristate 13-acetate (PMA)/0.5 .mu.M ionomycin(Io) (both from Sigma-Aldrich, St Louis, Mo.) were added in the presenceof 3 .mu.g/ml Brefeldin A (eBioscience, San Diego, Calif.) and, 6 hourslater, cells were harvested and analyzed for intracellular expression ofIL-10 and IFN-gamma. The non-activating doses of PMA and ionomycin usedin these experiments did not induce T-cell proliferation, but weresufficient to induce cytokine synthesis by T-cells. IL-10 levels weremeasured using an Alexa 488-conjugated anti-human IL-10 antibody(eBioscience, San Diego, Calif.) as described in Example 6. IFN-γ levelswere measured by FACS, using a PE-labeled anti-human IFN-γ antibody(eBioscience, San Diego, Calif.).

The results of this analysis are shown in FIGS. 16. More than 20% offreshly-isolated T-cells expressed IFN-γ, while less than 1% expressedIL-O, after suboptimal stimulation with PMA/ionomycin (i.e., of thecells that expressed either IFN-γ or IL-10, over 95% expressed IFN-γ andless than 5% expressed IL-10). However, after 7 days' co-culture witheither SB623 cells or MSCs, of the cells expressing either IFN-γ orIL-10, more than 95% expressed IL-10, while less than 5% expressedIFN-γ. Thus, co-culture with either MSCs or SB623 cells converted theT-cell secretome from one that was pro-inflammatory to one that wasanti-inflammatory.

The secretion of the inflammatory cytokine IFN-γ is a characteristic ofthe T_(H)1 subset of helper T-cells; while IL-10 secretion ischaracteristic of T_(H)2 cells and T_(reg) cells. Thus, the shift fromIFN-γ secretion to IL-10 secretion, observed upon co-culture of naiveT-cells with SB623 cells or MSCs, is consistent with conversion of apopulation rich in T_(H)1 cells into one that contains a large amount ofT_(H)2 cells, T_(reg) cells, or both. This result also indicates thatco-culture with SB623 cells, or MSCs, directed the differentiation ofT-cells from an inflammatory population (characterized by T_(H)1 cells)to an more anti-inflammatory population (characterized by T_(H)2 cellsand/or T_(reg) cells), in part through altering cytokine production bythe T-cells.

Example 8 Effect of MSC and SB623 Cell Co-Culture on Production of IL-17by T-Cells

Two of the cytokines known to be secreted by MSCs and SB623 cells. TGFβ-1 and IL-6 (see Example 3, above) are also known to play a role in thedevelopment of Th17 helper T-cells (i.e., helper T cells that secreteIL-17). Accordingly, T-cells were cultured in the presence of IL-23,which is known to stimulate the development of Th17 helper T-cells, andthe effect of co-culture with MSCs or SB623 cells, on Th17 cell number,was determined.

For these experiments, human T-cells were isolated and cultured asdescribed in Example 7, above, with the addition of 10 ng/ml of IL-23(Peprotech, Rocky Hill, N.J.) to the cultures. After treatment withBrefeldin A for 6 hours, cells were harvested, fixed and permeabilizedas described in Example 7, stained with a PE-conjugated anti-IL17antibody (eBioscience) and analyzed by flow cytometry. The resultsindicated that culture of T-cells in the presence of IL-23 increased thenumber of IL-17-expressing cells. In addition, co-culture of T-cellswith MSCs or SB623 cells resulted in a small increase in the number ofIL-17-expressing cells, in both the absence and presence of IL-23. (FIG.17).

Example 9: Inhibition of the Differentiation of Monocytes into DendriticCells by Co-Culture with SB623 Cells

The normal course of development of monocytes (expressing CD14) intodendritic cells (which express CD1a) can be recapitulated in vitro byculturing monocytes in the presence of interleukin-4 (IL-4) andgranulocyte/macrophage colony-stimulating factor (GM-CSF). MSCs, whenco-cultured with monocytes in vitro, are able to block thedifferentiation of monocytes into dendritic cells, an effect that ismediated, in part, by secretion of interleukin-6 (IL-6) by MSCs.Chomarat et al. (2000) Nature Immunology 1:510-514; Djouad et al. (2007)Stem Cells 25:2025-2032. SB623 cells also secrete IL-6. See U.S. PatentApplication Publication No. 2010/0266554 (Oct. 21, 2010). VEGF, which isalso secreted by MSCs and SB623 cells, is also involved in dendriticcell differentiation. Therefore, the effect of SB623 cells on monocytedifferentiation was investigated.

Peripheral blood was collected from healthy donors and subjected todensity gradient centrifugation using Ficoll-Paque™ (GE Healthcare.Piscataway, N.J.). Mononuclear cells were recovered by aspirating thebuffy coat, resuspended in RPMI/10% fetal bovine serum and plated. Afterovernight culture at 37.degree. C., 5% CO₂, non-adherent cells werewashed off and adherent monocytes were recovered using 0.25% trypsin/2mM EDTA. Staining with FITC-conjugated anti-human CD14 antibody (Becton,Dickinson & Co., San Jose, Calif.) indicated that over 90% of the cellsin these preparation were monocytes).

Monocytes were cultured in RPMI-1640 (Meidatech, Manassas, Va.)containing 10% fetal bovine serum (Lonza, Allendale, N.J.), 2 mML-glutamine, 2 mM L-sodium pyruvate, 100 Units/ml penicillin, 100 ug/mlstreptomycin, 40 ng/ml GM-CSF (Peprotech, Rocky Hill, N.J.) and 20 ng/mlIL-4 (Peprotech, Rocky Hill, N.J.). Co-culture with SB623 cells (orMSCs, as control) was conducted at a 10:1 ratio of monocytes to SB6323cells (or MSCs); i.e., 100,000 monocytes to 10,000 SB623 cells or MSCs.After 7 days of culture (or co-culture), a portion of the cells wereharvested using trypsin/EDTA (as above) and incubated with PE-conjugatedanti-CD 14 antibody and FITC-labeled anti-CD1a antibody (both fromeBioscience, San Diego, Calif.). Acquisition and analysis were performedusing a FACSCalibur™ cell sorter using CellQuestPro™ software (both fromBecton, Dickinson & Co., San Jose, Calif.). Another portion of thecultures were observed by phase-contrast microscopy.

The results of the cell sorting analysis (FIGS. 18A and 18B) indicated ahigher percentage of CD14⁺ cells (i.e., a higher fraction of monocytes)following co-culture of monocytes with SB623 cells or MSCs. Moreover,the effect was greater when monocytes were cultured with SB623 cells,compared to co-culture with MSCs. In addition, fewer CD1a⁺ dendriticcells were observed in the co-cultures. These results indicate thatSB623 cells (and, to a lesser extent, MSCs) are able to block thedifferentiation of monocytes into dendritic cells.

Microscopic analysis confirmed these observations. In monocyte cultures,clusters of dendritic cells were readily observed by microscopy; but inco-cultures with MSCs or SB623 cells, such clusters were rarelyobserved.

Example 10 Inhibition of Dendritic Cell Maturation by Co-Culture withSB623 Cells

After differentiating from monocytes, dendritic cells mature into a cellthat expresses the CD86 surface marker. This maturation can berecapitulated in vitro by culturing dendritic cells in the presence oftumor necrosis factor-alpha (TNF-α). IL-6 and VEGF have been shown toblock the maturation of dendritic cells. Park et al. (2004) J. Immunol.173:3844-3854; Takahashi et al. (2004) Cancer Immunol. Immunother53:543-550. Since SB623 cells secrete both of these cytokines, theeffect of SB623 co-culture on dendritic cell differentiation wasinvestigated.

To assess the effect of co-culture of SB623 cells on maturation ofdendritic cells, monocytes were obtained from peripheral blood anddifferentiated in vitro into dendritic cells, as described in Example 9.After 5 days of culture, human (TNF-α (Peprotech, Rocky Hill, N.J.) wasadded to the cultures to a final concentration of 10 ng/ml. In somecultures, SB623 cells or MSCs were also added at this time. All samplescontained 10⁵ monocytes and, in co-cultures, 10⁴ MSCs or SB623 cells. Asa control, Cyclosporin A, which inhibits maturation of dendritic cellsto a CD86⁺ state, was added to TNF-α-stimulated cultures to a finalconcentration of 1 ug/ml. Two days later, cells were stained withPE-conjugated anti-CD86 antibodies (Becton Dickinson & Co., San Jose,Calif.), acquired on a FACSCalibur™ cell sorter and analyzed usingCellQuest Pro™ software (both from Becton, Dickinson & Co., San Jose,Calif.).

The results, shown in FIG. 19, indicate that a significant fraction ofTNF-α-matured dendritic cells express CD86, and that this fraction islowered by treatment with Cyclosporine A, as expected. Co-culture withSB623 cells and MSCs also lowers the fraction of CD86⁺ cells. Notably,SB623 cells had a stronger inhibitory effect on dendritic cellmaturation, as measured by CD86 expression, than did MSCs.

Example 11 Alteration of the Secretory Profile of Monocytes/Macrophagesby Co-Culture with SB623 Cells

Human peripheral blood monocytes expressing the CD14 cell surface marker(i.e., macrophage precursors) were obtained from cells of the buffy coatby magnetic selection, using anti-CD14-coated magnetic beads (MiltenyiBiotec, Auburn, Calif.). Separate cultures of the CD 14⁺ monocytes wereexposed to granulocyte/macrophage colony-stimulating factor (GM-CSF),which converts them to M1 (pro-inflammatory) macrophages; or tomacrophage colony-stimulating factor (M-CSF), which converts them to M2(anti-inflammatory) macrophages; or were co-cultured with either SB623cells or MSCs.

The percentage of cells expressing tumor necrosis factor-alpha (TNF-α, apro-inflammatory cytokine characteristic of M1 macrophages) andinterleukin 10 (IL-10, an anti-inflammatory cytokine characteristic ofM2 macrophages) were determined in these cultures, as follows. Cultureswere exposed to 100 ng/ml bacterial lipopolysaccharide (LPS, Sigma, St.Louis, Mo.) for 24 hours. During the final 6 hours of exposure to LPS,Brefeldin A and monensin (both from eBioscience San Diego, Calif.; andboth used at 1:1,000 dilution) were added to the cultures. Cells werethen stained with either PE-conjugated anti-TNF-α or FITC-conjugatedanti-IL-10 and analyzed by flow cytometry.

The results of these studies, shown in FIG. 20A and 20B, indicated thatco-culture with MSCs or SB623 cells increased the fraction of monocytesin the culture that produced anti-inflammatory cytokines. Co-culturewith MSCs or SB623 cells did not increase the percentage of cells thatproduced TNF-α, as did exposure to GM-CSF (FIG. 20A). Notably, thepercentage of cells expressing the anti-inflammatory cytokine IL-10 wasincreased when monocytes were co-cultured with MSCs, and was increasedeven further when monocytes were co-cultured with SB623 cells (FIG.20B).

We claim:
 1. A method for suppressing a peripheral immune response in asubject, wherein the method comprises: administering to the subject aneffective amount of cells obtained by a method comprising: (a) providinga culture of marrow adherent stromal cells; (b) contacting the cellculture of step (a) with a polynucleotide comprising sequences encodinga Notch intracellular domain (NICD) wherein said polynucleotide does notencode a full-length Notch protein; (c) selecting cells that comprisethe polynucleotide of step (b); and (d) further culturing the selectedcells of step (c) in the absence of selection for the polynucleotide;wherein the cells of step (d) suppress the peripheral immune response byone or more of: (i) suppressing proliferation of naïve CD4⁺T-cells, (ii)suppressing activation of naïve CD4⁺T-cells, (iii) stimulatingproduction of regulatory T-cells (T_(reg)s), (iv) increasing productionof IL-10 by T-cells, (v) decreasing production of IFN-γ by T-cells, (vi)increasing production of IL-17 by T-cells, (vii) inhibiting thedifferentiation of monocytes into dendritic cells, (viii) inhibitingmaturation of dendritic cells, (ix) increasing production of IL-10 bymonocytes, (x) decreasing the relative concentration of T_(H)1 cells,and (xi) increasing the relative concentration of T_(H)2 cells.
 2. Themethod of claim 1, wherein the peripheral immune response results fromrheumatoid arthritis.
 3. The method of claim 1, wherein the peripheralimmune response results from an allogeneic transplantation.
 4. Themethod of claim 1, wherein the peripheral immune response results fromischemia.
 5. The method of claim 1, wherein the peripheral immuneresponse results from necrosis.
 6. The method of claim 1, wherein theperipheral immune response results from graft-versus-host disease(GVHD).
 7. The method of claim 1, wherein the peripheral immune responseresults from graft rejection.
 8. The method of claim 1, wherein theperipheral immune response results from an autoimmune disorder.
 9. Themethod of claim 8, wherein the autoimmune disorder is selected from thegroup consisting of multiple sclerosis, ulcerative colitis, chronicobstructive pulmonary disease (COPD), asthma, lupus and Type I diabetes.